490 BACTERIOPHAGES 



terium was not a function of the latent period for that particular 

 bacterium. 



The only technical difficulty in carrying" out such an experi- 

 ment is the distribution of the large number of accurately meas- 

 ured sam.ples into tubes within the latent period. This sampling is 

 greatly facilitated by an automatic pipetting machine, a number 

 of which are on the market. One very convenient device is the 

 Cornwall Pipetting Unit* with which one person can easily dis- 

 tribute 200 samples in 5 min. 



Host Cell Mutation to Resistance to Virus Attack 



If 10 ml. of a broth culture of strain B of E. coli is grown to a 

 concentration of 10^ cells/ml. and is then inoculated with coli 

 phage T6 to a concentration of 10^ particles/ml., the culture 

 will clear in a few hours owing to lysis of the susceptible bacteria. 

 However, if the culture is further incubated overnight it will 

 again become turbid owing to growth of phage-resistant variants 

 of strain B. 



Isolation of Phage- Resistant Mutants 



A phage-resistant mutant can usually be isolated by simply 

 spreading a mixture of host cells and virus on an agar plate, incu- 

 bating and picking surviving colonies. 



Example: One ml. of a bacterial culture containing- 2 X 10^ cells/ml. is 

 mixed with 1 ml. of T6 phage containing 10^" particles/ml., or an input ratio 

 of 50:1. After 5 min. incubation all bacteria which can adsorb phage are 

 infected. One-tenth ml. of the adsorption mixture is placed on the surface of 

 an agar plate and spread uniformly with a glass rod until all of the liquid has 

 been adsorbed by the agar. After incubation the colonies are counted and 

 12 are found. A single isolated colony is picked from this plate, suspended 

 in 1 ml. of broth and a loopful restreaked on another plate. Two repetitions 

 of this procedure should result in a pure strain of the variant which is free from 

 contaminating virus. If this strain is now plated with phage T6 by the agar 

 layer method, no plaques will be produced. Furthermore, phage T6 is not 

 adsorbed to this variant. 



* Made by Becton, Dickinson & Co., Rutherford, N. L 



